Association of Tumor Necrosis Factor-α(TNF-α) -308G>A and -238G>A Polymorphisms with Recurrent Pregnancy Loss Risk: A Meta-Analysis

Background Multiple studies have been carried out examining the association of tumor necrosis factor-α gene (TNF-α) promoter region polymorphisms with recurrent pregnancy loss (RPL) risk. However, the results remain con- troversial and incomplete. Hence, we performed a meta-analysis to evaluate the association of the TNF-α -308G>A and -238G>A polymorphisms with RPL risk. Materials and Methods In this meta-analysis, a comprehensive search of PubMed, Web of Knowledge and EM- BASE was performed to identify relevant studies published until December 1, 2017. The associations were assessed by odds ratio (OR) and its corresponding 95% confidence interval (CI). Results A total of 29 case-control studies, comprising 20 studies on TNF-α -308G>A (3,461 cases and 3,895 con- trols) and nine studies on TNF-α -238G>A (2,589 cases and 2,664 controls), were included in the meta-analysis. Over- all, we found TNF-α -308G>A to be associated with an increase in RPL risk under the homozygote (OR=1.716, 95% CI: 1.210-2.433, P=0.002) and the recessive (OR=1.554, 95% CI: 1.100-2.196, P=0.012) models. TNF-α -238G>A was also significantly associated with increased risk of RPL under the allele model (OR=1.554, 95% CI: 1.100-2.196, P=0.012). Stratified analysis revealed a more significant association between the TNF-α -308G>A polymorphism and increased RPL risk in Asians under the homozygote (OR=2.190, 95% CI: 1.465-3.274, P≤0.001), the dominant (OR=1.642, 95% CI: 1.269-2.125, P≤0.001) and the recessive (OR=1.456, 95% CI: 1.039-2.040, P=0.029) models, but not in Caucasians. A non-significant association was, however, identified between TNF-α -238G>A and RPL risk based on ethnicity. Moreover, TNF-α -308G>A and -238G>A polymorphisms were significantly associated with in- creased risk of RPL in high quality studies and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) subgroups. Conclusion The present meta-analysis demonstrates that TNF-α -308G>A and -238G>A polymorphisms are associ- ated with an increased risk of RPL.


Introduction
Recurrent pregnancy loss (RPL) is traditionally defined as the occurrence of three or more (≥3) consecutive pregnancy losses; however, the American Society of Reproductive Medicine (ASRM) has recently redefined RPL as two or more pregnancy losses (1,2). It is estimated that up to 3% of fertile couples have been diagnosed with RPL (3). Moreover, RPL is accompanied by an increased risk of other pregnancy complications such as preterm birth or small for gestational age newborns (4). RPL remains one of the most important issues in reproductive medicine and there are multiple barriers to the prevention, diagnosis and treatment of it (5).
TNF-α, a key pro-inflammatory cytokine, is secreted by macrophages and plays an important role in apoptotic cell death and initiating an immune response (10,11). TNF-α is located on human chromosomes 6p21.3, spanning 2,762bp, and contains 4 exons (11,12). There are several common single nucleotide polymorphisms (SNPs) in TNF-α which can regulate the transcription and production of TNF-α (12). To date, several promoter region SNPs in TNF-α have been reported, among which two, namely -308G>A (rs1800629) and -238G>A (rs361525), are most frequently studied in RPL (11)(12)(13). These polymorphisms have been shown to contribute to the susceptibility of several autoimmune conditions (9,12,13). Moreover, numerous studies have examined the association between TNF-α polymorphisms and risk of RPL; however, results have been controversial and inconclusive. These inconsistencies may be partly due to low sample sizes, false positive findings, publication bias, ethnic and geographical heterogeneity, and different characteristics among studies such as sources of controls. Meta-analysis is an important tool to obtain an unbiased estimate of the role of genetic variation in disease susceptibility (14). Thus, we conducted a meta-analysis to comprehensively evaluate the association of TNF-α -308G>A and -238G>A polymorphisms with susceptibility to RPL.

Search strategy
This meta-analysis was conducted and reported in accordance with the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines. All studies published up to December 1, 2017 reporting the association of the TNF-α -308G>A and -238G>A polymorphisms with RPL were identified by searching the literature in databases including PubMed, EMBASE, ISI Web of Science, Google Scholar, China National Knowledge Infrastructure (CNKI), and Wanfang. The following combination of MeSH terms and keywords was used: (''tumor necrosis factor alpha'' OR ''TNF-α'' OR ''cachexin'' OR ''cachectin'') AND (''-308G>A'' OR ''rs1800629'' OR ''-238G>A'' ''rs361525'') AND (''recurrent pregnancy loss'' OR ''pregnancy loss'' OR ''miscarriage'' OR ''RPL'' OR "habitual abortion'' OR ''abortion'' OR ''unexplained recurrent spontaneous abortion'') AND (''gene'' OR ''allele'' OR ''genotype'' OR ''mutation'' OR ''variant'' OR ''variation'' OR ''polymorphism''). In addition, the reference lists of retrieved articles, reviews and previous meta-analyses were manually screened for additional studies. In the case where more than one article was published by the same author using the same case series, the study with the largest sample size was investi-gated. Moreover, no restrictions were placed on language, and only published studies with full-text articles were included.

Inclusion and exclusion criteria
Studies fulfilling the following selection criteria were included in this meta-analysis: i. Has original and published data, ii. Uses case-control or cohort design, iii. Examines the associations of TNF-α -308G>A and -238G>A polymorphisms with RPL risk, and iv. Provides sufficient data for calculation of odds ratio (OR) with 95% confidence interval (CI). In addition, the following exclusion criteria were also used: i. Not relevant to TNF-α -308G>A and -238G>A polymorphisms and RPL, ii. The design is based on family or sibling pairs, iii. No usable data reported, iv. The study only involved a case population, v. Animal studies, vi. Duplicated publications, and vii. Abstracts, case-only articles, editorials, and reviews. If studies had partly overlapped subjects, only the one with the largest sample size was included.

Data extraction
Two investigators independently reviewed full manuscripts of eligible studies, and the relevant data were extracted into predesigned data collection forms. Any discrepancy was resolved by discussion or consensus by involving a third reviewer when required. The following data were collected from each study: first Authors' surname, year of publication, country of origin, ethnicity of the study population, genotyping method, source of control groups (population-or hospital-based controls), total number of cases and controls as well as numbers of cases and controls for each TNF-α SNP genotype, and deviation from Hardy-Weinberg Equilibrium (HWE) of the control group. Diverse ethnicities were categorized as Asian, African, Latinos and Caucasian.

Statistical analysis
The association of TNF-α -308G>A and -238G>A polymorphisms with RPL was estimated by ORs and their 95% CIs. Z-test was carried out to evaluate the statistical significance of pooled ORs (P<0.05 was considered statistically significant). The pooled ORs for both polymorphisms were performed under the following five genetic models: allele model (B vs. A), homozygote model (BB vs. AA), heterozygote model (BA vs. AA), dominant model (BA+BB vs. AA) and recessive model (BB vs. BA+AA). The heterogeneity of studies was assessed by using Cochran's Q-test and the I 2 test. A significance level of <0.10 was used to indicate heterogeneity among studies. Moreover, a high value of I 2 indicated a higher probability of the existence of heterogeneity (I 2 =0 to 25%, no heterogeneity; I 2 =25 to 50%, moderate heterogeneity; I 2 =50 to 75%, large heterogeneity; and I 2 =75 to 100%, extreme heterogeneity). When between-study heterogeneity was found a random-effects model was performed; otherwise, a fixed-effects model (Mantel-Haenszel method) was employed. HWE of genotype distribution in the controls of included studies was conducted using an online program (http://ihg2.helmholtzmuenchen.de/cgi-bin/hw/hwa1.pl), and P<0.05 was considered as significant deviation from HWE. Stratified analyses were performed according to ethnicity, source of controls and study quality (HWE status). To validate the reliability and stability of the results, sensitivity analysis was performed with a single study in the meta-analysis being removed each time to reflect the influence of the individual data set on the pooled OR. The funnel plot was employed to examine publication bias. Egger's regression analysis was used for re-evaluation of publication bias. The significance of the intercept was determined by the t test suggested by Egger, with P<0.10 considered as representative of statistically significant publication bias. Funnel plots and Egger's linear regression tests were used to provide a diagnosis of the potential publication bias. In the presence of bias, we utilized the Duval and Tweedie non-parametric ''trim and fill'' method to adjust results. The statistical analysis for this meta-analysis was performed by using the comprehensive meta-analysis (CMA) version 2.20 software (Biostat, USA) using two-sided P values.

Results
The search process and search outcomes are listed in Figure 1. One hundred and forty-six potential studies were collected by database and manual search. After screening titles and abstracts, 30 studies were considered duplicates and were excluded. Following the full-text review, 45 articles were excluded as they were reviews, case reports, not case-control designed, contained no data concerning RPL and two lacked any data concerning the polymorphisms of interest. Moreover, we also examined recent reviews and meta-analyses; no additional relevant study was found. Finally, 29 case-control studies in 20 publications with 5,050 RPL cases and 6,559 controls were included in the metaanalysis. There were 20 case-control studies (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34) for the TNF-α -308G>A polymorphism including 3,461 RPL cases and 3,895 controls, and nine case-control studies (25-28, 30-32, 34) for the TNF-α -238G>A polymorphism including 2,589 RPL cases and 2,664 controls (Table 1). Data from studies published between 2001 and 2017 were pooled to the meta-analysis. Among the 29 case-control studies, there were eight studies of Caucasians, 16 studies of Asians, two studies of Latinos and two studies of Africans. Studies had been carried out in United Kingdom, Brazil, Argentina, Germany, Iran, Mexico, Tunisia, Italy, China, Bahrain, India, Korea, and Saudi Arabia. The TNF-α polymorphisms were genotyped by five methods including polymerase chain reaction-amplification refractory mutation system (PCR-ARMS), PCR-restriction fragment length polymorphism (PCR-RFLP), sequence-specific oligonucleotide (SSO), PCR-sequence-specific primer (PCR-SSP) and direct sequencing. The distribution of genotypes in the controls of five studies (20, 26, 27, 32, 33) deviated from HWE expectations (P<0.05). The detailed characteristics of each study and genotype distributions included in the meta-analysis are presented in Table 1.  Table 2 lists the main results of the meta-analysis of TNF-α -308G>A polymorphism and RPL risk. When all the eligible studies were pooled into the meta-analysis, a significant association was found under the homozygote model (AA vs. GG: OR=1.716, 95% CI: 1.210-2.433, P=0.002, Fig.2A) and the recessive model (AA vs. AG+GG: OR=1.554, 95% CI: 1.100-2.196, P=0.012, Fig.2B). In addition, significant between-study heterogeneity was detected for all genetic models.

TNF-α-308G>A polymorphism
When stratified by ethnicity, a significant association with increased RPL risk was observed among Asians under the homozygote genetic model ( . Subgroup analysis of studies with high quality data did not show a significant association between this polymorphism and increased risk of RPL. Summary results of different comparisons are listed in Table 2.   Table 3 lists the main results of the meta-analysis of TNF-α -238G>A polymorphism and RPL risk. When all the eligible studies were pooled into the meta-analysis, a significant association was found under the allele model (A vs. G: OR=1.456, 95% CI: 1.039-2.040, P=0.029, Fig.2C). Interestingly, when stratified by ethnicity, there was no significant association with an increased RPL risk in the Asian population. Stratified analysis according to genotyping technique revealed a significantly increased risk of RPL in those studies involving PCR-RFLP under the allele model (A vs. G: OR=1.418, 95% CI: 1.077-1.867, P=0.013).

Test of heterogeneity and sensitivity analyses
There was significant heterogeneity among these studies for the TNF-α -308G>A polymorphism under the allele model (A vs. G: P H ≤0.001), the heterozygote model (AG vs. GG: P H =0.009) and the dominant model (AA+AG vs. GG: P H ≤0.001). Then, we assessed the source of heterogeneity by meta-regression analysis. Surprisingly, ethnicity, glaucoma types, genotyping methods and study quality did not contribute to substantial heterogeneity in the meta-analysis (Table 2).   Additionally, we performed sensitivity analysis to confirm the stability and reliability of our results by sequentially omitting individual eligible studies. When any single study was excluded, the corresponding ORs were not considerably changed, indicating the stability of the estimated OR. In addition, we excluded the studies in which genotype distribution in the controls deviated from HWE expectations, and in the homozygote and recessive models, the studies were found to affect the corresponding pooled ORs with heterogeneity removed under the recessive model ( Table 2). The supplementary sensitivity analysis thus showed that the results of the present meta-analysis are reliable.

Publication bias
We used the funnel plot and Egger's linear regression to assess publication bias of the included studies. The shapes of the funnel plots indicated no obvious asymmetry (Fig.3). The results of Egger's test also showed no strong statistical evidence of publication bias ( Table 2). However, Egger's test found evidence for the publication bias between TNF-α -308G>A polymorphism and RPL risk under the homozygote model (AA vs. GG: P Begg's =0.273, P Egger's =0.046, Fig.3B). Therefore, we used the Duval and Tweedie non-parametric ''trim and fill'' method to adjust for publication bias. However, the results with and without ''trim and fill'' did not lead to different conclusions, indicating that our results are statistically robust.

Discussion
Although RPL is one of the most common public health issues, little is known regarding genetic its susceptibility factors (35). Genetic variants in TNF-α have been associated with the etiology of RPL (9). Several important polymorphisms in the promoter region of TNF-α have been identified, such as the -308G>A and -238 G>A variants (27)(28)(29)(30)(31)(32)(33)(34). Hitherto, the associations between polymorphisms in the promoter region of TNF-α and the risk of RPL have remained inconclusive. Thus, we performed a CMA to evaluate the association of TNF-α -308G>A polymorphisms with risk of RPL. The meta-analysis found a significant association between the TNF-α -308G>A polymorphism and RPL under the homozygote and re-cessive models. Moreover, to the best of our knowledge, this meta-analysis is the first to evaluate the association of TNF-α -238G>A polymorphism with RPL. The results of our meta-analysis revealed that the TNF-α -238G>A polymorphism was associated with the increased risk of RPL under the allele model. However, due to the limited number of available published studies on the TNF-α -238G>A polymorphism, further studies with larger sample sizes are needed to reach a more convincing conclusion.
The subgroup analyses revealed a significant association between TNF-α -308G>A polymorphism and RPL in Asian populations under the three genetic models. However, for Caucasians, the results indicated that this polymorphism is not associated with increased risk of RPL. The inconsistency of subgroup analysis with pooled estimates may be due to genetic diversity in different ethnicities. Furthermore, given that RPL is a multifactorial condition, beside genetic factors, endogenous and exogenous factors play a major role in RPL aetiology. Similarly, the included studies in this meta-analysis differed in their findings with regard to the association between TNF-α -308G>A polymorphism and risk of RPL. It is therefore possible that the difference between the studies may reflect the different ethnicities been investigated, because geographical regions may have different genetic and environmental factors that might affect the findings. Thus, this discrepancy might also be due to other factors such as maternal cigarette smoking, caffeine consumption, alcohol consumption, maternal age, number of previous miscarriages, diabetes mellitus, infective agents, endocrine factors, uterine anatomic abnormalities and antiphospholipid antibody syndrome (APS). Therefore, the relationship between this polymorphism and RPL might vary by ethnicity.
Recently, a meta-analysis by Li et al. (9) with 1,430 RPL cases and 1,727 healthy controls was performed to investigate the associations of TNF-α polymorphisms with RPL. Their results suggested that the TNF-α -308G>A polymorphism was associated with increased RPL risk. Our results are consistent with their meta-analysis. However, their meta-analysis generated contradictory results due to insufficient power because the number of studies was considerably smaller than that needed to achieve robust conclusions. In addition, due to small size, they could not rule out the possibility that publication bias was undetected. More recently, two new epidemiological studies (33,34) have been performed to estimate the effect of the TNF-α -308G>A polymorphism on RPL risk in Asian populations. Moreover, we found that Li et al. had overlooked seven studies on the TNF-α -308G>A polymorphism and RPL risk in their meta-analysis. Hence, it may significantly affect their total results and RPL results. In the present meta-analysis, by including more nine casecontrol studies, we found that the TNF-α -308G>A polymorphism was associated with risk of RPL.
Between-study heterogeneity and publication bias are important issues that cannot be ignored in a meta-analysis (36,37). In addition, between-study heterogeneity might distort the conclusion of a meta-analysis (38). The study designs, source of controls subjects, ethnicity, genotyping method, sample size and other variables may contribute to the heterogeneity (37)(38)(39)(40). Here, we detected moderate between-study heterogeneity across studies under the allele, heterozygote and the dominant models. We thus selected the random-effects model to summarize the ORs. We performed meta-regression analysis to find the source of between-study heterogeneity. However, after the subgroup analysis by ethnicity, heterogeneity still remained in Caucasians. In contrast, in the Asians, there were no heterogeneity, indicating that heterogeneity could be partly accounted for by the genetic distribution of different ethnicities between continents. We observed publication bias for the association between the TNF-α -308G>A polymorphism and RPL risk under the homozygote model. After subgroup analysis by ethnicity, the publication bias disappeared in both Caucasians and Asians.
This meta-analysis has a number of limitations that should be noted. First, we could not perform further subgroup analysis by ethnicity among Caucasians and other ethnicities because of the limited number of published studies. Secondly, we strictly followed the inclusion and exclusion criteria to reduce possible selection bias. However, the Egger's linear regression test showed little publication bias of overall analysis under the homozygote genetic model. Thirdly, the sources of literature searched were from a limited selection of electronic databases, and we failed to retrieve unpublished studies and articles written in other languages and also unpublished studies that might meet the inclusion criteria. Thus, potentially, publication bias might exist even though the funnel plots were found to be symmetrical. Fourthly, subgroup analyses regarding age, number of miscarriage, and other risk factors such as chronic disease were not conducted since the primary literature lacked sufficient data. Finally, the TNF-α -308G>A and -238G>A polymorphisms were not analyzed in combination with other polymorphisms of TNF-α and the effect of gene-gene interactions on RPL development was not undertaken. The relationship between polymorphisms of TNF-α gene and other genetic and environmental risk factors may be highly complicated, and extensive research is still required to ascertain how exactly the TNF-α polymorphisms affect the susceptibility of an individual to RPL.

Conclusion
This meta-analysis confirmed the association of the TNF-α -308G>A and -238G>A promoter region polymorphisms with increased risk of RPL. Moreover, our metaanalysis suggests that TNF-α -308G>A is more likely to be associated with the risk of RPL in Asians than Caucasians. Further large, well-designed case-control studies are needed to confirm these findings.